PROJECT 3: PROJECT SUMMARY/ABSTRACT A major clinical problem in the management of prostate cancer is the difficulty associated with treating aggressive cancers, especially those that are highly castration resistant. The androgen signaling pathway remains a key therapeutic target for advanced prostate cancer but resistance to agents targeting this pathway is common, highlighting the need to develop novel therapeutic approaches. Mouse prostate cancer modeling has elucidated molecular pathways of aggressive, castration-resistant prostate cancer (CRPC) which include loss of the tumor suppressors PTEN and TP53 and overexpression of the MYC oncogene. Using these spontaneous mouse models of prostate cancer we have identified Ephrin receptor EphB4 as a potential therapeutic target. EPHB4 is a receptor tyrosine kinase that with its ligand ephrin B2, are not expressed in normal prostate gland, but are expressed in a majority of human prostate cancers. EPHB4 is induced by multiple pathways important for CRPC development, including loss of PTEN and TP53 as well as activation of the PI3K pathway downstream of EGFR and IGF1R. In turn, EPHB4 activation engages multiple signaling pathways, including the PI3 kinase/AKT and MAPK pathways known to modulate the androgen receptor and drive CRPC development. To test the significance of EphB4, we generated conditional EphB4 knockout mouse. We found that genetic deletion of EphB4 or its inhibition using a soluble antagonist (sEPHB4) profoundly inhibited prostate tumorigenesis driven by loss of Pten and led to the regression of established tumors in transgenic mice. This was associated with inhibition of PI3K/AKT signaling and apoptosis. Notably, sEpBh4 antagonist and EphB4 knockdown led to markedly lower levels of androgen receptor (AR) protein. These functional genetic data lead us to hypothesize that EPHB4 is a novel pharmacologic target with high therapeutic potential in prostate cancer, including CRPC. We will explore this hypothesis by targeting EphB4 in genetically complex mouse models (loss of Pten, Tp53 and Myc over-expression) and human xenograft models of prostate cancer and CRPC, singly or in combination with AR-targeted therapy (including enzalutamide, abiraterone). We will examine human prostate tumor samples including metastases and CRPCs for the expression of EphB4, EphrinB2, and downstream markers. A soluble decoy EPHB4 receptor ? human serum albumin fusion protein (sEPHB4HSA) antagonist is in early human trials in other tumors, and has been found to be remarkably safe in Phase I study. We will therefore implement a feasibility clinical trial of sEPHB4HSA aimed at determining the therapeutic efficacy of targeting EPHB4 in men with CRPC. Successful completion of the preclinical and early clinical studies we propose in this application could lead to a rapid translation of soluble EPHB4 antagonist as a treatment for advanced prostate cancer.